This invention relates generally to the field of microfluidic devices, such as are used for analysis of various biological and chemical compositions. Generally, the invention relates to separation of particulate matter from liquid samples. In a preferred embodiment, the invention relates to a method and apparatus for separating samples of blood for analysis of its components.
To determine the presence (or absence) of, or the amount of an analyte, such as glucose, albumin, or bacteria in bodily or other fluids, a reagent device is generally used to assist a technician performing the analysis. Such reagent devices contain one or more reagent areas at which the technician can apply the sample fluid and then compare the result to a standard. For example, a reagent strip is dipped into the sample fluid and the strip changes color, the intensity or type of color being compared with a standard reference color chart. Preparation of such devices is difficult when the sample has a complex composition, as many bodily fluids do. The component to be identified or measured may have to be converted to a suitable form before it can be detected by a reagent to provide a characteristic color. Other components in the sample fluid may interfere with the desired reaction and they must be separated from the sample or their effect neutralized. Sometimes, the reagent components are incompatible with each other. In other cases, the sample must be pre-treated to concentrate the component of interest. These and other problems make it difficult to provide in a single device the reagent components which are needed for a particular assay. The art contains many examples of devices intended to overcome such problems and to provide the ability to analyze a fluid sample for a particular component or components.
A different approach is to carry out a sequence of steps which prepare and analyze a sample, but without requiring a technician to do so. One way of doing this is by preparing a device which does the desired processes automatically, but by keeping the reagents isolated, is able to avoid the problems just discussed.
Carrying out analysis may involve receiving a sample, selecting a desired amount of the sample, diluting or washing the sample, separating it into components, and carrying out reactions with the sample or its components. If one were to carry out such steps in a laboratory on large samples, it would generally be necessary for a technician to manually perform the necessary steps or if automated, equipment would be needed to move the sample and its components and to introduce reagents, wash liquids, diluents and the like. However, it is typical of biological assays that the samples are small and therefore it follows that the processing steps must be carried out in very small equipment. Scaling down laboratory equipment to the size needed for samples of about 0.02 to 10.0 μL is not feasible and a different approach is used. Small vessels connected by μm size passageways are made by creating such features in plastic or other suitable substrates and covering the resulting substrate with another layer. The vessels may contain reagents added to them before the covering layer is applied. The passageways may also be treated as desired to make them wettable or non-wettable by the sample to be tested. The sample, its components, or other fluids may move through such passageways by capillary action when the walls are wetted or they are prevented from moving when the fluids do not wet the walls of the passageway. Thus, the capillary sized passageways can either move fluids or prevent their movement as if a valve were present. Another method of moving fluids through such μm sized passageways is by centrifugal force, which overcomes the resistance of non-wettable walls. This simple description provides an overview of microfluidic devices. Specific applications are provided in many patents, some of which will be mentioned below.
An extended discussion of some of the principles used in arranging the vessels and passageways for various types of analyses is provided in U.S. Pat. No. 6,143,248 and additional examples of applications of those principles may be found in U.S. Pat. No. 6,063,589. The microfluidic devices described in those two patents were intended to be disposed in disc form and rotated on equipment capable of providing varying degrees of centrifugal force as needed to move fluids from one vessel to another. Generally, a sample would be supplied close to the center of rotation and gradually increasing rotational speeds would be used to move the sample, or portions of it, into vessels disposed further away from the center of rotation. The patents describe how specific amounts of samples can be isolated for analysis, how the samples can be mixed with other fluids for washing or other purposes, and how samples can be separated into their components.
Other patents describe the use of electrodes for moving fluids by electro-osmosis, such as U.S. Pat. No. 4,908,112. Caliper Technology Corporation has a portfolio of patent on microfluidic devices in which fluids are moved by electromotive propulsion. Representative examples are U.S. Pat. No. 5,942,443; 5,965,001 and 5,976,336.
In U.S. Pat. No. 5,141,868 capillary action is used to draw a sample into a cavity where measurements of the sample can be made by electrodes positioned in the sample cavity.
Whole blood is often separated into its components for medical uses or for analysis. Since the components of blood have different specific gravities, the red blood cells (RBC) being the heaviest and plasma being the lightest, separation is usually done by subjecting the blood to high centrifugal forces. According to the American Association of Blood Banks, separation of whole blood into red blood cells and platelet rich plasma requires application of 2,000 times the force of gravity for 3 minutes, typically in centrifuges designed to separate whole blood in the plastic bags in which the blood was collected.
Separation of red blood cells and plasma can also be done using devices which use filters to block the passage of the red blood cells while allowing the plasma to pass through. One example is found in U.S. Pat. No. 4,600,507 in which a tube for mounting in a centrifuge has a filter aligned along the axis of the tube. In WO 01/24931 a sample is filtered and the liquid passing through the filter is transferred via capillary passages. A weir serves as a filter in the design disclosed by Yuen et al in Genome Research 11:405–412, 2001. Guigan in U.S. Pat. No. 4,788,154 describes a complex device for use in separating small amounts of blood using centrifugation. The device is turned 180° on a turntable during the separation process. In published U.S. Patent Application 2001/0046453 A1 blood is not separated, but the blood samples are combined with a reagent, and then flow by capillary forces into a waste well without the use of centrifugation.
One potential use for microfluidic devices is the analysis of blood samples. However, the small size of the passageways in such devices has been reported to result in blockage by blood cells. The present inventors have found this to be a problem when the passageways are less than about 60 μm in size. Furthermore, in some methods of analysis, the blood components should be analyzed separately. For example, the red blood cells may interfere with colorimetric analysis of clear plasma. Therefore, it is often important that blood samples be separated so that the components can be analyzed and that blockage of the small passageways in microfluidic devices can be avoided. One example of the use of microfluidics to separate blood into its fractions is found in U.S. Pat. No. 6,063,589 discussed above. A rather complex arrangement of elements is used, which are generally larger than those of the present invention. Relatively large rotational speeds are used to separate whole blood into its fractions. The patent does not provide information on the surface properties of the fluidic elements. Although the effect of wetting or non-wetting surfaces is mentioned. Similarly, in U.S. Pat. No. 5,160,702 a complex arrangement of channels and chambers is shown in which samples of blood are separated and analyzed. A curved separating chamber is used having one end spaced radially further from the center of rotation than the opposite end.
In the invention to be described below, a microfluidic device is shown which is configured to provide rapid separation of red blood cells and plasma with minimal centrifugal force.